The present invention relates generally to isolation and utilization of novel proteins, and more particularly to a unique receptor-signaling-complex component referred to as the tumor necrosis factor receptor associated factor interacting protein.
Members of the TNFR (Tumor Necrosis Factor Receptor) superfamily play important roles in the induction of diverse signals leading to cell growth, activation and apoptosis. Smith et al., Cell, 76:959-962 (1994). Whether the signals induced by a given receptor leads to a cell""s activation or death is highly cell-type specific and tightly regulated during differentiation of the cell. For example, the TNFRs can exert co-stimulatory signals for proliferation of naive lymphocytes, but can also induce death signals required for deletion of activated T lymphocytes. Smith et al., Cell, Id.
The cytoplasmic domains of these receptors lack intrinsic catalytic activity and generally exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNF-R1 which share significant homology within an 80 amino acid region of their cyto-plasmic tails, referred to in the art as the xe2x80x9cdeath domain.xe2x80x9d Tartaglia et al., Cell, 74:845-853 (1993); Itoh et al., J. Biol. Chem. 268:10932-10937 (1993). Therefore, the TNFR family members are believed to initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex. Smith et al., Cell, Id.
Several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily. Rothe et al., Cell, 78:681-692 (1994); Cheng et al., Science, 267:1494-1498 (1995); Nakano et al., J. of Biol. Chem., 271: 14661-14664 (1996). Recent biochemical and molecular studies show that a class of signal transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains. Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J. Biol. Chem., 270:7795-7798 (1995); Hsu et al., Cell, 81:495-504, (1995). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD(MORT1)/RIP through the interactions of their respective death domains. Itoh et al., J. Biol. Chem., Id.; Tartaglia et al., Cell, Id.; Hsu et al., Immunity, 4:387-396 (1996). The clustering of these signal transducers leads to the recruitment of FLICE/MACH, and subsequently, to cell death. Muzio et al., Cell, 85:817-827 (1996); Boldin et al., Cell, 81: 803-815 (1996).
The TNFR family members can also recruit a second class of signal transducers called TRAF (Tumor necrosis factor Receptor Associated Factor), some of which are responsible for the activation of NF-kB or JNK. Hsu et al., Cell, 84:299-308 (1996); Liu et al., Cell, 87:565-576. TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30 or LT-xcex2R receptors which interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails but which do not interact with the death domain containing proteins. Sato et al., FEBS Letters, 358:113-118 (1995); Song et al., Biochem. J., 809:825-829 (1995); Lee et al., J. Exp. Med., 183:669-674 (1996).
Distinct members of the TPAF family have been identified as signaling components of the TNFR family. All TRAF members contain a conserved TRAF domain, approximately 230 amino acids in length, that is used for either homo- or hetero-oligomerization among the TRAF family to interact with the cytoplasmic regions of the TNFRs or for interactions with downstream signal transducers. Rothe et al., Proc. Natl. Acad. Sci. USA, 93:8241-8246 (1996); Song et al., Proc. Natl. Acad. Sci. USA, 93:6721-6725 (1996); Cheng et al., Genes Dev., 10:963-973 (1996). In addition to the TRAF domain, most of the TRAF family members contain an N-terminal RING finger and several zinc finger structures which appear to be important for their effector functions. Regnier et al., J. of Biol. Chem., 270:25715-25721 (1995); Hu et al., J. Biol. Chem., 269:30069-30072 (1994); Moisalos et al., Cell, 80: 389-399 (1995).
Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2, was subsequently shown to mediate NF-kB activation induced by two TNF receptors, CD40 and CD30. Rothe et al., Science, 269:1424-1427 (1995); Lee et al., Proc. Natl. Acad. Sci. USA, 93: 9699-9703 (1996). TRAF5 was also implicated in NF-kB activation mediated by LT-bR, whereas TRAF3 (also known as CRAF1, CD40bp or LAP1) was shown to be involved in the regulation of CD40-mediated CD23 up-regulation in B cells. Cheng et al., Science, Id. Other TRAF members in the TNFR family-mediated signal transduction have potential effector functions as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the c-IAP (cellular Inhibitor of Aoptosis Protein) family to the TNFR2 signaling complex. Rothe et al., Cell, 83:1243-1252 (1995).
In addition to signal transduction with TNFR family members, TRAFs also have the potential to regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of interleukin-1 receptor (IL-1R) signaling complex in which it mediates the activation of NF-kB by IL-1R. Cao et al., Nature, 383:443-446 (1996). Since TRAFs form homo- or hetero-oligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers.
Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, there is a need in the art for a determination as to whether TRAF proteins will also mediate the apoptotic signals induced by the death-domain-less members of the TNFR superfamily. Zheng et al., Nature, 377:348-351 (1995); Gruss et al., Blood, 83: 2045-2056 (1994); Amakawa et al., Cell, 84:551-562 (1996).
In view therefore, the present disclosure describes the isolation and characterization of a novel protein component that associates with the receptor-TRAF signaling complex and inhibits the TRAF2-mediated NF-kB activation, which can determine whether a given cell proliferates or dies.
In accordance with the present invention, a novel component of the receptor-TRAF signaling complex, designated TRIP (Tumor necrosis factor Receptor associated factor Interacting Protein) has been identified, characterized and disclosed uniquely herein. The TRIP structure contains a RING finger motif and an extended coiled-coil domain, and when associated with the TNFR2 or CD30 signaling complex through TRAF transducers, TRIP functions as an inhibitor of TRAF2-mediated NF-kB activation. Since TRAF2-mediated NF-kB activation is closely linked to prevention of cellular apoptosis, TRIP, its active fragments, its structural or functional analogs, and its agonists or antagonists, can all be used as receptor-proximal regulators for influencing signals responsible for cell activation/proliferation or cell death.
Stimulation of members of the TNFR superfamily activates signaling cascades leading to the regulation of a cell""s activation, growth or death. Many of these signal transducers contain either TRAF or death domains, which mediate protein-protein interactions. The TRAF family proteins interact directly with some members of the TNFR family and play a role in the activation of signaling pathways induced by these receptors. Once associated with the receptors, these proteins recruit downstream signal molecules that act to initiate cascades leading to cell activation or death. The present invention includes the identification and characterization of a novel regulator proximal to the TNFR/TRAF signaling complex.
The structure of the TRIP includes an N-terminal RING finger motif followed by a long coiled-coil domain divided into two subdomains. Amino acid sequences of the N-terminal half of the coiled-coil domain of TRIP shows about 50% similarity to the rod-like, coiled-coil structure of the myosin heavy chain, while those of the C-terminal half of the coiled-coil domain are characteristic of a leucine-zipper.
The coiled-coil domain of TRIP is required, not only for TRIP-TRAF interactions, but also for the inhibition of TRAF2-mediated NF-KB activation by TRIP. The RING finger domain of TRIP plays a regulatory role based on analogy to other RING-finger proteins. The C-terminal half of TRIP distal to the coiled-coil domain does not show any significant homology to other proteins but contains several phosphorylation sites, suggesting kinase regulation of TRIP.
TRIP is recruited to the receptors, TNFR2 or CD30, via its interaction with TRAF proteins. The recruitment of TRIP to these receptors is efficient in the presence of the TRAF2 oligomer. TRIP also inhibits the induction of NF-kB activation mediated by TNFR1, which indirectly interacts with TRAF2 via TRADD (see page 2, supra). In transient transfection assays, TRIP inhibited NF-kB activation induced by TNFR2, CD30 and TNFR1, and also by TRADD, all of which activates NF-kB via TRAF2. However, TRIP did not inhibit the activation of NF-kB by IL-1R which is mediated by TRAF6, confirming that the negative effect of TRIP on NF-kB activation is specific to a TRAF2-mediated pathway.
The specificity demonstrated by TRIP makes it unique among other signal transducers such as I-TRAF and A20 which inhibit TRAF2-mediated NF-kB activation. In contrast to TRIP, both I-TRAF and A20 inhibit the activation of NF-kB induced by IL-1R as well as by TNFRs. TRIP differs from I-TRAF or A20 in several additional aspects. First, TRIP is recruited to the cognate receptor-TRAF signaling complex, while I-TRAF is not; and TRIP can be recruited to the cognate receptors via its interaction with TRAF2 homo-oligomer, while A20 interacts only with TRAF2-TRAF1 hetero-oligomer.
Second, the inhibitory mechanism acting on NF-kB activation by I-TRAF, A20 and TRIP appears to be different. I-TRAF inhibits TRAF2-mediated NF-kB activation by blocking the recruitment of TRAF2 to the receptor complex which would normally initiate the clustering of TRAF proteins. In contrast, TRIP is recruited to the receptor complex by its association with TRAF2. Although A20 interacts with TRAFs, its inhibitory effect on TRAF2-mediated NF-kB activation does not require direct protein-protein interaction in transfection assays. TRIP, however, inhibits TRAF2-mediated NF-kB activation only when its coiled-coil domain, required for the TRIP-TRAF interaction, is intact.
TRIP specificity is also demonstrated in contrast with the c-IAPs (see pages 3-4, supra). TRIP and the c-IAP""s are the only two protein types which are recruited to the receptor-TRAF complex. In contrast to TRIP, however, c-IAPs do not exert a negative effect on the activation of NF-kB induced by receptors. In addition to their functional differences, TRIP and c-IAPs are recruited differently to their cognate receptors. TRIP can be recruited to the cognate receptors (TNFR2 or CD30) in the presence of TRAF2 homo-oligomer, while c-IAPs are recruited to TNFR2 only through TRAF2-TRAF1 hetero-oligomer.
As a receptor-proximal negative regulator of NF-kB activation, TRIP through signals mediated by the TNFR2- or CD30-TRAF signaling complex can initiate seemingly opposing effects on cells, namely cell activation/growth or cell death. The balance of pro-activation/growth or pro-cell death signals mediated by the receptor-TRAF complex are controlled by the particular set of signal transducers (i.e. c-IAPs or TRIP) which are recruited to the receptor complex. When c-IAPs are recruited to the receptor complex, TRAF2-mediated NF-kB activation proceeds unaffected. The activation of NF-kB induces the expression of various genes and also suppresses cell death which drives the cells towards the pro-activation/growth state. When TRIP is recruited to the receptor complex it inhibits NF-kB activation which is required for anti-apoptotic signals. In addition, the contributions of other anti-apoptotics such as manganese superoxide dismutase or A20 will be diminished. Thus, the particular signals from the receptor-TRAF-TRIP complex will drive cells toward the anti-activation, pro-cell death state.
TRIP is further characterized by its particular effects on lymphoctes. The choice of which type of signal transducer (c-IAPs or TRIP) is to be recruited to the cognate receptors is most likely determined by their availability and by the presence of different TRAF proteins such as TRAF1. The expression of TRAF1 is tissue-specific while that of TRAF2 is not, and when lymphocytes are stimulated to proliferate via their antigen receptors the expression of c-IAP1 or TRAF1 is upregulated while TRIP expression is decreased. Consistently, TRAF2 expression is not significantly affected during lymphocyte proliferation. During antigen-stimulation of lymphocytes, therefore, the formation of TRAF2-TRAF1-c-IAP complex will be favored and recruited to the cognate TNFR family members, which exerts co-stimulatory signals for lymphocyte proliferation. TRAF1 overexpression antagonizes the formation of TRAF2 homo-oligomer in cells, which inhibits the activation-induced cell death of mature CD8+ T cells, normally mediated by the TNFR2 signaling complex. TRIP expression is also most abundant in thymocytes which are destined to die during clonal deletion which is in part mediated by CD30.
In one aspect, the present invention extends to a novel protein having the following characteristics:
1. A structure which contains an extended coiled-coil domain, in particular, a domain selected from the amino acid residue sequences numbered 56-275 shown in FIGS. 2A-2B (SEQ ID NO: 3) (SEQ ID NO: 4); and
2. which, when associated with the TNFR2 or CD30 signaling complex through TRAF transducers, functions as an inhibitor of TRAF2-mediated NF-kB activation.
In a further aspect, the invention comprises TRIP, in particular, TRIP having an amino acid sequence selected from the full-length sequences shown in FIGS. 2A-2B (SEQ ID NO: 1) (SEQ ID NO: 2), its active fragments, its structural or functional analogs, and its agonists or antagonists, which can all be used as receptor-proximal regulators for influencing signals responsible for cell activation/proliferation or cell death.
TRIP can be characterized as a novel regulator proximal to the TNFR/TRAF signaling complex which has in its protein structure (FIG. 2C):
1. an N-terminal RING finger motif, in particular, those having an amino acid sequence selected from the sequences shown in FIG. 2D, SEQ ID NO: 5 and SEQ ID NO: 6;
2. followed by a long coiled-coil domain divided into two subdomains, the first subdomain being similar to the myosin heavy chain domain, and the second C-terminal subdomain characterized by a leucine-zipper.
Functionally, TRIP is recruited to TNFR2 or CD30 receptors via interaction with TRAF proteins, specifically the TRAF2 homo-oligomer. TRIP inhibits the induction of NF-kB activation induced by TNFR2, CD30, TNFR1 and TRADD specifically through the TRAF2-mediated pathway. TRIP does not inhibit the activation of NF-kB by IL-1R which is mediated by TRAF6.
TRIP inhibits TRAF2-mediated NF-kB activation only when its coiled-coil domain, required for the TRIP-TRAF interaction, is intact. The inhibition via the receptor-TRAF-TRIP complex drives an effected cell toward the anti-activation, pro-cell death state. When lymphocytes are stimulated to proliferate via their antigen receptors, the expression of TRIP is decreased, while TRIP expression is abundant in thymocytes which are destined to die.
The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a nucleic acid molecule, in particular, a recombinant DNA molecule or cloned gene, which has a nucleotide sequence selected from the sequences shown in FIG. 8A (SEQ ID NO: 7) and FIG. 8B (SEQ ID NO: 8). According to other features of certain embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human TRIP, its active fragments, and its structural or functional analogs.
The human and murine DNA sequences (SEQ ID NO: 7) (SEQ ID NO: 8) for TRIP or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for TRIP. The present invention also includes the preparation of plasmid vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIG. 8A (SEQ ID NO: 7) and FIG. 8B (SEQ ID NO: 8). Correspondingly, the preparation of antisense RNA and ribozymes are included herein.
In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding TRIP, and more particularly, the complete DNA sequences shown in FIG. 8A (SEQ ID NO: 7) and FIG. 8B (SEQ ID NO: 8).
The concept of the present invention contemplates that specific factors exist for correspondingly specific ligands, such as for the TNFR2, CD30, TNFR1 and TRADD and the like which have a specificity for the TRAF2-mediated pathway, as described earlier. Accordingly, this specificity and the direct involvement thereto by TRIP offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
The present invention naturally contemplates several means for preparation of TRIP, its active fragments, and its structural or functional analogs, including known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the cDNA (SEQ ID NO: 7) (SEQ ID NO: 8) and amino acid sequences disclosed herein facilitates their reproduction by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human TRIP.
The invention includes an assay system for screening of potential drugs effective to modulate the activity of target mammalian cells by interrupting or potentiating the effects of TRIP, its active fragments, and its structural or functional analogs.
The assay system could be adapted to identify drugs or other entities that are capable of binding to TRIP, its active fragments or its structural/functional analogs, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating NF-kB activity. Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity. For example, such drugs might be used to inhibit lymphocyte apoptosis thereby prolonging the cellular life of certain sub-populations of T cells.
In yet a further embodiment, the invention contemplates antagonists of the activity of TRIP. In particular, an agent or molecule which promotes TRAF2-mediated NF-kB activation. In a specific embodiment, the antagonist can be a peptide having a sequence complementary to the coiled-coil domains as shown by amino acid residues 56-275 shown in FIG. 2A.
The present invention also extends to the development of antibodies against TRIP, its active fragments, and its structural or functional analogs, including naturally raised and recombinantly prepared antibodies. For example, the anti-bodies could be used to screen expression libraries to obtain the gene or genes that encode for TRIP in other animal species. Or such anti-bodies could be used to diagnose for TRIP deficiency or overexpression in potential human patients.
Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating TRIP activity.
Thus, TRIP, its active fragments, its structural or functional analogs, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the aforementioned proteins that has been labeled by either radioactive addition, or radioiodination.
In an immunoassay, a control quantity of antibodies to TRIP, or the like, may be prepared and labeled with an enzyme a specific binding partner and/or a radio-active element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectro-photometric, amperometric or gasometric techniques known in the art.
The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of TRIP, its active fragments, its structural or functional analogs, or to identify drugs or other agents that may mimic or block their activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein which couples a label to a binding partner for TRIP such as an anti-TRIP antibody, and one or more additional immunochemical reagents.
In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of TRIP, its subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of TRIP or its subunits, and comprises administering an agent capable of modulating the production and/or activity of TRIP or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. For example, drugs or other binding partners to TRIP may be administered to inhibit or potentiate NF-kB activity.
More specifically, the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of activation of TRIP or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention. For example, drugs or other binding partners to TRIP or proteins as represented by SEQ ID NOS: 1-6, may be administered to inhibit or potentiate TRIP activity.
Accordingly, it is a principal object of the present invention to provide the TRIP protein and its subunits in purified form that exhibits certain characteristics and activities associated with cell growth, activation, proliferation and apoptosis.
It is a further object of the present invention to provide antibodies to the TRIP and its subunits, and methods for their preparation, including recombinant means.
It is a further object of the present invention to provide a method for detecting the presence of TRIP and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse effects of the TRIP and/or its subunits in mammals.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of TRIP or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of TRIP or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the TRIP, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the TRIP.
Yet a further object is to provide a test kit for the quantitative analysis of the extent of the presence of TRIP, its active fragments, its structural or functional analogs, or to identify drugs or other agents that may mimic or block their activity.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.